This proposal aims to assess the interaction of T cells with dendritic cells (DC) under conditions of tolerance and activation. The lab has set up an excellent model to evaluate the response of auto-reactive CD4+ T cells upon recognition of self-antigen. In this model DO11.10 CD4+ T cells specific for OVA peptide (323-339) are transferred into transgenic mice, which express OVA as a systemically secreted soluble protein (sOVA-Tg). Previous studies in the lab have shown that in a "full" sOVA-Tg host, the auto-reactive T cells become tolerant, while in an "empty" or lymphopoenic sOVA-TgRag-/- host, T cells respond with uncontrolled activation and host mice develop autoimmunity. The strengths of this model include the ability to track single cells specific for a true self-antigen during tolerance, and well-defined conditions for breaking tolerance, resulting in activation and autoimmunity allowing us to directly compare these responses. Our central hypothesis is that there is competition for space or survival factors when self-reactive T cells see their antigen in the presence of endogenous T cells, resulting in tolerance. We propose to define whether tolerance can be visualized during the initial interaction with DC's presenting self-antigen, or whether it is a result of defective regulation later on. We will use cutting edge technology including two-photon microscopy to determine if there are "stop" signals upon initial encounter of T cells with self-antigen. Donor cells will be labeled with a cell tracker dye and host DC's will be marked by using CD11c-EYFP mice crossed to the sOVA-Tg mice. We will then be able to evaluate the localization, kinetics, movement and duration of interactions of the initial encounter of T cells with DC's under tolerance or activation. We will assess the influence of competition by co-transferring polyclonal T or B cells in this system. It has been demonstrated, that the activation status of the DC can subsequently define the T cell response, primarily through its ability to relay information to the responding T cell. We will address the activation status of the DC's under conditions of tolerance using a combination of whole mount immunohistochemistry and Quantitative RT-PCR. We will address whether activation signals such as CpG-oligonucleotides or anti-CD40 signaling are enough to change the status of the DC's and break tolerance. Understanding the interactions of self-reactive T cells with DC's presenting self-antigen is key to understanding the induction of tolerance vs. immunity. How this balance is maintained and what causes disregulation, resulting in autoimmunity, is a vital question in immunology today, and integral for the design of therapies in the future.